Advanced pancreatic ductal adenocarcinoma (PDAC) is often inoperable, and is only transiently responsive to existing therapies. Overexpression of indoleamine 2,3-dioxygenase (IDO) in PDAC plays a major role in accelerating disease progression by suppressing antitumor immunity. Current IDO inhibitors inadequately reverse immunosuppression while systemic off target effects contribute to their toxicity. ShIDO-ST is a novel Salmonella typhimurium (ST)-based therapy that expresses a small hairpin (sh)RNA to specifically silence tumor-derived IDO with decreased toxicity. ST as an shRNA delivery vehicle offers superior penetration against desmoplastic PDAC tissue and anti-metastatic function due to its motility and affinity for poorly vascularized, hypoxic tissue. he combination of IDO silencing and abundant ST tumor colonization increases intratumoral local reactive oxygen species through the recruitment and activation of polymorphonuclear neutrophils (PMN), which can cause oxidative stress-induced apoptosis of tumor cells, cancer stem cells (CSC), and vascular stroma to inhibit PDAC progression. PMN are also known to have anti-metastatic function, thus providing additional protection against tumor spread. The proposed work will provide detailed analysis of signaling pathways influencing PMN recruitment and activation upon shIDO-ST treatment. Cytokines involved in PMN recruitment will be evaluated using protein array analysis and flow cytometry of PDAC tumors from mice treated with shIDO-ST versus scrambled sequence control (shScr-ST), which does not silence IDO. The polarization and apoptotic state of infiltrating PMN will also be evaluated using flow cytometry, as IDO activity is known to regulate PMN function. The finding that both ST and PMN provide primary and anti-metastatic protection supports evaluation of shIDO-ST in more aggressive models of PDAC. The KrasG12D;Trp53R127H;Pdx1-Cre genetically engineered mouse model (KPC-GEMM) recapitulates desmoplasia and metastasis characteristic of human PDAC. Exclusive access to fresh surgical PDAC tissue, by virtue of our close collaboration with surgical oncologists, will allow for evaluation of human (Hu)shIDO-ST in xenograft models to provide greater support for clinical translation. Combination therapy with Sutent(tm) (Pfizer), which induces vascular collapse in PDAC, will be tested because it increases the hypoxic area of the tumor to augment colonization by ST and the recruitment of PMN. To improve penetration of tumors by shIDO-ST and Sutent, we propose using PEGylated human recombinant hyaluronidase (PEGPH20, Halozyme Inc.), which depletes hyaluronan abundant in PDAC tissue and increases vascular permeability. The effects of proposed treatments on primary and metastatic burden, CSC frequency, and ST colonization will be evaluated in transgenic and xenograft settings through cross sectional or longitudinal measurements of tumor volume, metastatic burden, CSC markers, and bacterial enumeration. The long-term objective is to develop shIDO-ST into a suitable, effective therapy for the treatment of patients with advanced inoperable PDAC.